Bursaphelenchus xylophilus detection and analysis system based on CRISPR - Cas12

Abstract

Pine wilt disease is caused by the pine wood nematode (Bursaphelenchus xylophilus) and leads to wilting and death of pines. It is one of the most damaging diseases of pines worldwide. Therefore, accurate and rapid detection methods are of great importance for the control of B. xylophilus. Traditional detection methods have some problems, such as being time-consuming and requiring expensive instruments. In this study, the loop-mediated isothermal amplification (LAMP) and clustered regularly interspaced short palindromic repeats (CRISPR) were used to establish a set of intelligent detection and analysis system for B. xylophilus, called LAMP-CRISPR/Cas12a analysis, which integrated field sampling, rapid detection and intelligent control analysis. The process can be completed within 1 hour, from sample pretreatment and detection to data analysis. Compared with the single LAMP method, the LAMP-CRISPR/Cas12a assay uses species-specific fluorescence cleavage to detect target amplicons. This process confirms the amplicon identity, thereby avoiding false-positive results from non-specific amplicons, and the large amounts of irrelevant background DNA do not interfere with the reaction. The LAMP-CRISPR/Cas12a assay was applied to 46 pine wood samples and the samples carrying B. xylophilus nematodes were successfully identified. To meet the needs of different environments, we designed three methods to interpret the data: 1) naked eye interpretation; 2) lateral flow biosensor assay; and 3) integrated molecular analysis system to standardize and intellectualize the detection process. Application of the B. xylophilus detection and analysis system will reduce the professional and technical requirements for the operating environment and operators and help to ensure the accuracy of the detection results, which is important in grass-root B. xylophilus detection institutions.

Keywords: Bursaphelenchus xylophilus; CRISPR-Cas12a; LAMP; nucleic acid detection; rapid diagnostic.

Figure 1

(A) Schematic illustration of loop-mediated isothermal amplification (LAMP). (B) Schematic illustration of CRISPR-Cas12a detection. Step 1: LAMP products are obtained. Protospacer adjacent motif (PAM) sites guide the CRISPR/Cas12a-gRNA complex to recognize target sites. Step 2: Cas12a effectors are activated. Step 3: The activated effectors nonspecifically cleave single-stranded DNA reporter molecules by trans-cleavage. (C) Schematic illustration of the LAMP-CRISPR/Cas12a assay workflow. The LAMP-CRISPR/Cas12a assay involves three closely linked steps: rapid template preparation (step 1), LAMP reaction (step 2), and CRISPR-Cas12a cleavage and signal detection (step 3).

Figure 2

(A) Fluorescence changes of three crRNA reactions for 1 h detected by CRISPR-Cas12a fluorescence assays. Values are shown in the graph as means ± SD (n = 3). (B) Fluorescence curves at different reaction temperatures. (C) Fluorescence curves of different crRNA concentrations. (D) Effects of different LAMP reaction times on the Cas12a reaction. NTC: no template control.

Figure 3

Specificity of the CRISPR-Cas12a enhanced fluorescent assay evaluated by its ability to distinguish base mismatches. Protospacer adjacent motif (PAM) sequences are shown in red; base mismatches are shown in green.

Figure 4

(A) Specificity of the LAMP-CRISPR/Cas12a assay evaluated by its ability to distinguish B xylophilus strains and related species. B.C: Botrytis cinerea; B.M: B mucronatus; QY: B xylophilus (from Liaoning); NJ: B xylophilus (from Nanjing); CQ: B xylophilus (from Chongqing); HS: B xylophilus (from Anhui); B.D: B doui; NTC: negative control (ddH2O). (B) End-point fluorescence visualization of the specificity test. (C) Detection of base changes or deletions in target sequences by LAMP-CRISPR/Cas12a assay by measuring fluorescence intensity under blue-light irradiation 1 h after the reaction. Panel 1: From left to right, 0–5 base changes; the last tube is the negative control. At four base changes, the fluorescence intensity dropped significantly. Panel 2: From left to right, 0–5 base deletions. the last tube is the negative control. At five base deletions, the fluorescence intensity dropped significantly.

Figure 5

(A) Sensitivity of the LAMP-CRISPR/Cas12a assay for B xylophilus detection. Purified B xylophilus genomic DNA was diluted by 10⁻¹, 10⁻², 10⁻³, 10⁻⁴, 10⁻⁵, 10⁻⁶, 10⁻⁷, and 10⁻⁸ to give DNA concentrations of 66.4 ng/µL, 6.64 ng/µL, 664 pg/µL, 66.4 pg/µL, 6.64 pg/µL, 0.664 pg/µL, 66.4 fg/µL, and 6.64 fg/µL. Different concentrations of DNA were detected by LAMP-CRISPR/Cas12a assay and visualized by blue light. (B) Results of the LAMP-CRISPR/Cas12a assay visualized by lateral flow biosensor. (C) Real-time fluorescence profiles of DNA at different concentrations in LAMP-CRISPR/Cas12a assay. (D) Sensitivity of the PCR assay for B xylophilus detection. The image shows amplification bands targeting the SYG-2 gene. Lanes 1–6 lanes are B xylophilus genomic DNA dilutions 664 ng/μL, 66.4 ng/µL, 6.64 ng/µL, 664 pg/µL, 66.4 pg/µL, and 6.64 pg/µL (3 replicates per concentration). (E, F) Different concentrations of DNA were detected by traditional LAMP and visualized by HNB and SYBRgreenI. (G) Real-time fluorescence curves of DNA with different concentrations in LAMP.

Figure 6

Processing scheme for the LAMP-CRISPR/Cas12a assay includes the following steps: DNA extraction, isothermal amplification reactions, and CRISPR-Cas12 detection of the B xylophilus by fluorescence.